Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the gradual loss of midbrain dopaminergic neurons in association with aggregation of α-synuclein. Oxidative damage has been widely implicated in this disease, though the mechanisms involved remain elusive. Here, we demonstrated that preferential accumulation of peroxidized phospholipids and loss of the antioxidant enzyme glutathione peroxidase 4 (GPX4) were responsible for vulnerability of midbrain dopaminergic neurons and progressive motor dysfunctions in a mouse model of PD. We also established a mechanism wherein iron-induced dopamine oxidation modified GPX4, thereby rendering it amenable to degradation via the ubiquitin-proteasome pathway. In conclusion, this study unraveled what we believe to be a novel pathway for dopaminergic neuron degeneration during PD pathogenesis, driven by dopamine-induced loss of antioxidant GPX4 activity.
Jie Sun, Xiao-Min Lin, Dan-Hua Lu, Meng Wang, Kun Li, Sheng-Rong Li, Zheng-Qiu Li, Cheng-Jun Zhu, Zhi-Min Zhang, Chang-Yu Yan, Ming-Hai Pan, Hai-Biao Gong, Jing-Cheng Feng, Yun-Feng Cao, Feng Huang, Wan-Yang Sun, Hiroshi Kurihara, Yi-Fang Li, Wen-Jun Duan, Gen-Long Jiao, Li Zhang, Rong-Rong He
Targeted therapies such as venetoclax (Bcl-2 inhibitor) have revolutionized the treatment of chronic lymphocytic leukemia (CLL). We previously reported that persister CLL cells in treated patients overexpress multiple anti-apoptotic proteins and display resistance to pro-apoptotic agents. Here, we demonstrated that multidrug resistant CLL cells in vivo exhibit apoptosis restriction at a premitochondrial level due to insufficient activation of the Bax and Bak proteins. Co-immunoprecipitation analyses with selective BH-domain antagonists revealed that the pleotropic pro-apoptotic protein (Bim) is prevented from activating Bax/Bak by “switching” interactions to other upregulated anti-apoptotic proteins (Mcl-1/Bcl-xL/Bcl-2). Hence, treatments that bypass Bax/Bak restriction are required to deplete these resistant cells in patients. Protein Phosphatase 2A (PP2A) contributes to oncogenesis and treatment resistance. We observed that a small molecule activator of PP2A (SMAP) induced cytotoxicity in multiple cancer cell lines and CLL samples, including multidrug resistant leukemia/lymphoma cells. The SMAP (DT-061) activated apoptosis in multidrug resistant CLL cells through induction of mitochondrial permeability transition pores (mPTP), independent of Bax/Bak. DT-061 inhibited the growth of wild type and Bax/Bak double knockout multidrug resistant CLL cells in a xenograft mouse model. Collectively, we discovered multidrug resistant CLL cells in patients, and validated a pharmacologically tractable pathway to deplete this reservoir.
Kallesh D. Jayappa, Brian Tran, Vicki L. Gordon, Christopher G. Morris, Shekhar Saha, Caroline C. Farrington, Caitlin M. O’Connor, Kaitin P. Zawacki, Krista M. Isaac, Mark Kester, Timothy P. Bender, Michael E. Williams, Craig A. Portell, Michael J. Weber, Goutham Narla
How cancer cells evade the therapeutic effects of immune checkpoint blockade is largely unknown. Here, we report that fibrinogen-like protein 1 (FGL1), a newly identified immune checkpoint ligand, was modified by acetylation at Lys 98 in hepatocellular carcinoma (HCC), which targeted it for proteasomal degradation. Sirtuin 2 (SIRT2) deacetylated and stabilized FGL1, thus promoting immune evasion. Notably, the SIRT2 inhibitor 2-Cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2) enhanced acetylation of FGL1 and reduced FGL1 protein levels in vitro. The combination of AGK2 and programmed death ligand 1 (PD-L1) blockade effectively suppressed tumor growth and improved overall survival of mice. Furthermore, aspirin, an old drug, could directly acetylate FGL1 at Lys 98 and promote its degradation in vitro. Aspirin enhanced the immunotherapeutic efficacy, induced tumor regression, and extended the lifespan of tumor-bearing mice. Furthermore, the SIRT2/FGL1 axis was expressed in HCC specimens. Collectively, these findings unveil an acetylation-mediated regulation of FGL1, identify a potential target for HCC immunotherapy, and provide therapeutic strategies for the clinical treatment of HCC.
Mingen Lin, Jing He, Xinchao Zhang, Xue Sun, Wenjing Dong, Ruonan Zhang, Yanping Xu, Lei Lv
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination (HR) repair proteins in 20-25% of cases. Defects in HR impart to tumor cells a specific vulnerability to poly-ADP ribose polymerase inhibitors and platinum-containing chemotherapy. However, not all patients who receive these therapies respond, and many who initially respond ultimately develop resistance. Inactivation of the HR pathway is associated with the overexpression of polymerase theta (Polθ, or POLQ). This key enzyme regulates the microhomology-mediated end-joining (MMEJ) pathway of double-strand break (DSB) repair. Using human and murine HR-deficient PDAC models, we find that POLQ knockdown is synthetically lethal with mutations in HR genes (BRCA1 and BRCA2) and the DNA damage repair gene ATM. Further, POLQ knockdown enhances cytosolic micronuclei formation and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling, leading to enhanced infiltration of activated CD8+ T cells in BRCA2-deficient PDAC tumors in vivo. Overall, POLQ, a key mediator in the MMEJ pathway, is critical for DSB repair in BRCA2-deficient PDAC. Its inhibition represents a synthetic lethal approach to block tumor growth while simultaneously stimulating an immune response.
Grace Oh, Annie Wang, Lidong Wang, Jiufeng Li, Gregor Werba, Daniel Weissinger, Ende Zhao, Surajit Dhara, Rosmel E. Hernandez, Amanda Ackermann, Sarina Porcella, Despoina Kalfakakou, Igor Dolgalev, Emily A. Kawaler, Talia Golan, Theodore H. Welling, Agnel Sfeir, Diane M. Simeone
Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call CerTra syndrome. These findings uncover a central role for CERT autoregulation in the control of the sphingolipid biosynthetic flux, provide unexpected insight into the structural organisation of CERT, and suggest a possible therapeutic approach for CerTra syndrome patients.
Charlotte Gehin, Museer A. Lone, Winston Lee, Laura Capolupo, Sylvia Ho, Adekemi M. Adeyemi, Erica H. Gerkes, Alexander P.A. Stegmann, Estrella López-Martín, Eva Bermejo-Sánchez, Beatriz Martínez-Delgado, Christiane Zweier, Cornelia Kraus, Bernt Popp, Vincent Strehlow, Daniel Gräfe, Ina Knerr, Eppie R. Jones, Stefano Zamuner, Luciano A. Abriata, Vidya Kunnathully, Brandon E. Moeller, Anthony Vocat, Samuel Rommelaere, Jean-Philippe Bocquete, Evelyne Ruchti, Greta Limoni, Marine Van Campenhoudt, Samuel Bourgeat, Petra Henklein, Christian Gilissen, Bregje W. van Bon, Rolph Pfundt, Marjolein H. Willemsen, Jolanda H. Schieving, Emanuela Leonardi, Fiorenza Soli, Alessandra Murgia, Hui Guo, Qiumeng Zhang, Kun Xia, Christina R. Fagerberg, Christoph P. Beier, Martin J. Larsen, Irene Valenzuela, Paula Fernández-Álvarez, Shiyi Xiong, Robert Śmigiel, Vanesa López-González, Lluís Armengol, Manuela Morleo, Angelo Selicorni, Annalaura Torella, Moira Blyth, Nicola S. Cooper, Valerie Wilson, Renske Oegema, Yvan Herenger, Aurore Garde, Ange-Line Bruel, Frederic Tran Mau-Them, Alexis B.R. Maddocks, Jennifer M. Bain, Musadiq A. Bhat, Gregory Costain, Peter Kannu, Ashish Marwaha, Neena L. Champaigne, Michael J. Friez, Ellen B. Richardson, Vykuntaraju K. Gowda, Varunvenkat M. Srinivasan, Yask Gupta, Tze Y. Lim, Simone Sanna-Cherchi, Bruno Lemaitre, Toshiyuki Yamaji, Kentaro Hanada, John E. Burke, Ana Marija Jakšić, Brian D. McCabe, Paolo De Los Rios, Thorsten Hornemann, Giovanni D'Angelo, Vincenzo A Gennarino
CRISPR-Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ leading to decreased plasmid stability secondary to induction of the cytidine deaminase APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFN-stimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies of inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR-Cas9 therapeutic approaches.
Mrinal K. Sarkar, Ranjitha Uppala, Chang Zeng, Allison C. Billi, Lam C. Tsoi, Austin Kidder, Xianying Xing, Bethany E. Perez White, Shuai Shao, Olesya Plazyo, Sirisha Sirobhushanam, Enze Xing, Yanyun Jiang, Katherine A. Gallagher, John J. Voorhees, J. Michelle Kahlenberg, Johann E. Gudjonsson
The origin of breast cancer, whether primary or recurrent, is unknown. Here, we show that invasive breast cancer cells exposed to hypoxia release small extracellular vesicles (sEV) that disrupt the differentiation of normal mammary epithelia, expand stem and luminal progenitor cells, and induce atypical ductal hyperplasia and intraepithelial neoplasia. This was accompanied by systemic immunosuppression with increased myeloid cell release of the “alarmin”, S100A9, and oncogenic traits of EMT, angiogenesis, and local and disseminated luminal cell invasion, in vivo. In the presence of a mammary gland driver oncogene (MMTV-PyMT), hypoxic sEV accelerated bilateral breast cancer onset and progression. Mechanistically, genetic or pharmacologic targeting of hypoxia-inducible factor-1α (HIF1α) packaged in hypoxic sEV, or homozygous deletion of S100A9 normalized mammary gland differentiation, restored T cell function and prevented atypical hyperplasia. The transcriptome of sEV-induced mammary gland lesions resembled luminal breast cancer, and detection of HIF1α in plasma circulating sEV from luminal breast cancer patients correlated with disease recurrence. Therefore, sEV-HIF1α signaling drives both local and systemic mechanisms of mammary gland transformation at high risk for evolution to multifocal breast cancer. This pathway may provide a readily accessible biomarker of luminal breast cancer progression.
Irene Bertolini, Michela Perego, Yulia Nefedova, Cindy Lin, Andrew T. Milcarek, Peter Vogel, Jagadish C. Ghosh, Andrew V. Kossenkov, Dario C. Altieri
Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.
Nafisa Nuzhat, Kristof Van Schil, Sandra Liakopoulos, Miriam Bauwens, Alfredo Dueñas Rey, Stephan Käseberg, Melanie Jäger, Jason R. Willer, Jennifer Winter, Hanh M. Truong, Nuria Gruartmoner, Mattias Van Heetvelde, Joachim C. Wolf, Robert Merget, Sabine Grasshoff-Derr, Jo Van Dorpe, Anne Hoorens, Heidi Stöhr, Luke Mansard, Anne-Françoise Roux, Thomas Langmann, Katharina Dannhausen, David Rosenkranz, Karl M. Wissing, Michel Van Lint, Heidi Rossmann, Friederike Häuser, Peter Nürnberg, Holger Thiele, Ulrich Zechner, Jillian N. Pearring, Elfride De Baere, Hanno J. Bolz
Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+ (NaV) and L-type Ca2+ channels (LTCC). LTCC dysregulation by mutant CaMs has been implicated in action potential duration (APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating NaV dysregulation within transverse (T)-tubules in D96V-CaM-associated arrhythmias. D96V-CaM induces pro-arrhythmic late Na+ current (INa) by impairing inactivation of NaV1.6, but not the predominant cardiac NaV isoform, NaV1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Super-resolution microscopy revealed close proximity of NaV1.6 and RyR2 within T-tubules. NaV1.6 density within these regions increased in cD96V relative to WT. Consistent with NaV1.6 dysregulation by D96V-CaM in these regions, we observed increased late NaV activity in T-tubules. The resulting late INa promoted aberrant Ca2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia (VT) in vivo. Cardiac-specific NaV1.6 knockout protected cD96V mice from increased T-tubular late NaV activity, and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promotes arrhythmias by dysregulating LTCC and NaV1.6 within T-tubules and thereby, facilitating aberrant Ca2+ release.
Mikhail Tarasov, Heather L. Struckman, Yusuf Olgar, Alec Miller, Mustafa Demirtas, Vladimir Bogdanov, Radmila Terentyeva, Andrew M. Soltisz, Xiaolei Meng, Dennison Min, Galina Sakuta, Izabella Dunlap, Antonia D. Duran, Mark P. Foster, Jonathan P. Davis, Dmitry Terentyev, Sándor Györke, Rengasayee Veeraraghavan, Przemysław B. Radwański
Although protein hydroxylation is a relatively poorly characterized post-translational modification, it has received significant recent attention following seminal work uncovering its role in oxygen sensing and hypoxia biology. Although the fundamental importance of protein hydroxylases in biology is becoming clear, the biochemical targets and cellular functions often remain enigmatic. JMJD5 is a ‘JmjC-only’ protein hydroxylase that is essential for murine embryonic development and viability. However, no germline variants in JmjC-only hydroxylases, including JMJD5, have yet been described that are associated with any human pathology. Here we demonstrate that biallelic germline JMJD5 pathogenic variants are deleterious to JMJD5 mRNA splicing, protein stability, and hydroxylase activity, resulting in a human developmental disorder characterised by severe failure to thrive, intellectual disability, and facial dysmorphism. We show that the underlying cellular phenotype is associated with increased DNA replication stress and that this is critically dependent on the protein hydroxylase activity of JMJD5. This work contributes to our growing understanding of the role and importance of protein hydroxylases in human development and disease.
Sally C. Fletcher, Charlotte L. Hall, Tristan J. Kennedy, Sander Pajusalu, Monica H. Wojcik, Uncaar Boora, Chan Li, Kaisa Teele Oja, Eline Hendrix, Christian A.E. Westrip, Regina Andrijes, Sonia K. Piasecka, Mansi Singh, Mohammed E. El-Asrag, Anetta Ptasinska, Vallo Tillmann, Martin R. Higgs, Deanna Alexis Carere, Andrew D. Beggs, John Pappas, Rachel Rabin, Stephen J. Smerdon, Grant S. Stewart, Katrin Õunap, Mathew L. Coleman
No posts were found with this tag.